The analysis of heart rate in the frequency domain has become increasingly important in physiological studies, and supports the use of heart rate variability as an index of autonomic cardiovascular control. A new index, the instant centre frequency (ICF) has been proposed as a global index of the instantaneous relationship between sympathetic and vagal modulation. The aim of this study was to assess ICF, RR intervals, and heart rate variability measures as indices of sympathovagal balance during a pharmacological blockade of the autonomic nervous system in normotensive rats. RR intervals and arterial blood pressure of 10 conscious Wistar rats equipped with telemetry probes, were evaluated before, during, and after injection of: (1) saline (100 microl kg(-1) i.v.); (2) phentolamine (5 mg kg(-1) i.v.); (3) atropine methyl nitrate (0.5 mg kg(-1) i.v.); and (4) atenolol (1 mg kg(-1) i.v.). RR interval series were analysed by the smoothed pseudo-Wigner-Ville distribution. A general linearised model was used to evaluate the parameters. ICF was calculated in the same way as the peak power frequency by use of the first moment of instant spectrum. We calculated the ICF of the whole spectrum (ICF(T)), ICF in high frequency (ICF(H)) and ICF in low frequency (ICF(L)). The RR intervals and ICF indexes varied similarly and presented the lowest coefficient of variation among animals exposed to the same autonomic conditions. ICF(T)-ICF(L) and ICF(H)-ICF(T) were strongly correlated with normalised HF and normalised LF. In normotensive rats, RR intervals and ICF indices may reliably capture the effects of the sympathetic and parasympathetic nervous system on the sinus node.